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• W2023973334 abstract "To investigate the exact biochemical functions by which Bcl-2 regulates apoptosis, we established a stable human small cell lung carcinoma cell line, Ms-1, overexpressing wild-type human Bcl-2 or various deletion and point mutants thereof, and examined the effect of these Bcl-2 mutants on apoptosis induced by antitumor drugs such as camptothecin. Cytochrome c release, caspase-3-(-like) protease activation, and apoptosis induced by antitumor drugs were accelerated by overexpression of Bcl-2 lacking a Bcl-2 homology (BH) 1 domain (Bcl-2/ ΔBH1), but not by that of BH2, BH3, or BH4 domain-deleted Bcl-2. A similar result was obtained upon the substitution of glycine 145 with alanine in the BH1 domain (Bcl-2/G145A), which failed to interact with either Bax or Bak. Pro-apoptotic Bax and Bak have been known to be activated in response to antitumor drugs, and Bcl-2/G145A as well as Bcl-2/ΔBH1 also accelerated Bax- or Bak-induced apoptosis in HEK293T cells. These two mutants still retained the ability to interact with wild-type Bcl-2 and Bcl-xL, and abrogated the inhibitory effect of wild-type Bcl-2 or Bcl-xL on Bax- or Bak-induced apoptosis. In addition, immunoprecipitation studies revealed that Bcl-2/ΔBH1 and Bcl-2/G145A interrupted the association between wild-type Bcl-2 and Bax/Bak. Taken together, our results demonstrate that Bcl-2/ΔBH1 or Bcl-2/G145A acts as a dominant negative of endogenous anti-apoptotic proteins such as Bcl-2 and Bcl-xL, thereby enhancing antitumor drug-induced apoptosis, and that this dominant negative activity requires both a failure of interaction with Bax and Bak through the BH1 domain of Bcl-2 and retention of the ability to interact with Bcl-2 and Bcl-xL. To investigate the exact biochemical functions by which Bcl-2 regulates apoptosis, we established a stable human small cell lung carcinoma cell line, Ms-1, overexpressing wild-type human Bcl-2 or various deletion and point mutants thereof, and examined the effect of these Bcl-2 mutants on apoptosis induced by antitumor drugs such as camptothecin. Cytochrome c release, caspase-3-(-like) protease activation, and apoptosis induced by antitumor drugs were accelerated by overexpression of Bcl-2 lacking a Bcl-2 homology (BH) 1 domain (Bcl-2/ ΔBH1), but not by that of BH2, BH3, or BH4 domain-deleted Bcl-2. A similar result was obtained upon the substitution of glycine 145 with alanine in the BH1 domain (Bcl-2/G145A), which failed to interact with either Bax or Bak. Pro-apoptotic Bax and Bak have been known to be activated in response to antitumor drugs, and Bcl-2/G145A as well as Bcl-2/ΔBH1 also accelerated Bax- or Bak-induced apoptosis in HEK293T cells. These two mutants still retained the ability to interact with wild-type Bcl-2 and Bcl-xL, and abrogated the inhibitory effect of wild-type Bcl-2 or Bcl-xL on Bax- or Bak-induced apoptosis. In addition, immunoprecipitation studies revealed that Bcl-2/ΔBH1 and Bcl-2/G145A interrupted the association between wild-type Bcl-2 and Bax/Bak. Taken together, our results demonstrate that Bcl-2/ΔBH1 or Bcl-2/G145A acts as a dominant negative of endogenous anti-apoptotic proteins such as Bcl-2 and Bcl-xL, thereby enhancing antitumor drug-induced apoptosis, and that this dominant negative activity requires both a failure of interaction with Bax and Bak through the BH1 domain of Bcl-2 and retention of the ability to interact with Bcl-2 and Bcl-xL. Apoptosis, a morphologically distinguished form of pro-grammed cell death, is critical not only during development and tissue homeostasis but also in the pathogenesis of a variety of diseases including cancer, autoimmune disease, and neuro-degenerative disorders (1Martin S.J. Green D.R. Crit. Rev. Oncol. Hematol. 1995; 18: 137-153Crossref PubMed Scopus (157) Google Scholar, 2Thompson C.B. Science. 1995; 267: 1456-1462Crossref PubMed Scopus (6205) Google Scholar, 3White E. Genes Dev. 1996; 10: 1-15Crossref PubMed Scopus (1325) Google Scholar). Mitochondria is the crucial regulatory organelle for the signaling pathway of apoptosis (4Desagher S. Martinou J.C. Trends Cell Biol. 2000; 10: 369-377Abstract Full Text Full Text PDF PubMed Scopus (1696) Google Scholar, 5Wang X. Genes Dev. 2001; 15: 2922-2933Crossref PubMed Scopus (94) Google Scholar). Many death signals cause irreversible dysfunction of mitochondria, leading to the release of several mitochondrial intermembrane proteins. Cytochrome c was the first characterized mitochondrial factor shown to be released from the mitochondrial intermembrane space and to be involved in the activation of caspase by forming apoptosomes (6Liu X. Kim C.N. Yang J. Jemmerson R. Wang X. Cell. 1996; 86: 147-157Abstract Full Text Full Text PDF PubMed Scopus (4484) Google Scholar, 7Zou H. Henzel W.J. Liu X. Lutschg A. Wang X. Cell. 1997; 90: 405-413Abstract Full Text Full Text PDF PubMed Scopus (2746) Google Scholar, 8Li P. Nijhawan D. Budihardjo I. Srinivasula S.M. Ahmad M. Alnemri E.S. Wang X. Cell. 1997; 91: 479-489Abstract Full Text Full Text PDF PubMed Scopus (6261) Google Scholar). Like cytochrome c, other mitochondrial intermembrane proteins such as AIF, Smac/DIABLO, Endo G, and Omi/HtrA2 were found to undergo release during apoptosis and have been implicated in various aspects of the cell death process (9Van Loo G. Saelens X. Van Gurp M. MacFarlane M. Martin S.J. Vandenabeele P. Cell Death Differ. 2002; 9: 1031-1042Crossref PubMed Scopus (532) Google Scholar). The Bcl-2 family of proteins plays a pivotal role in the regulation of mitochondrial integrity and response to apoptotic signals (10Wang H-G. Reed J.C. Histol. Histopathol. 1998; 13: 521-530PubMed Google Scholar). Bcl-2 family proteins can be subdivided into three distinct groups: (i) anti-apoptotic members such as Bcl-2 and Bcl-xL with sequence homology at Bcl-2 homology 1 (BH1), 1The abbreviations used are: BH1, Bcl-2 homology 1; SCLC, small cell lung carcinoma; PARP, poly(ADP-ribose) polymerase; wt, wild-type; PI, propidium iodide; EGFP, enhanced green fluorescent protein. BH2, BH3, and BH4 domains; (ii) pro-apoptotic molecules, such as Bax and Bak, with sequence homology at BH1, BH2, and BH3; and (iii) pro-apoptotic proteins that share homology only at the BH3 domain, such as Bid, Bik, Noxa, and Bim (11Gross A. McDonnell J.M. Korsmeyer S.J. Genes Dev. 1999; 13: 1899-1911Crossref PubMed Scopus (3268) Google Scholar). These Bcl-2 family members are characterized by their ability to interact and form homo- and heterodimers (10Wang H-G. Reed J.C. Histol. Histopathol. 1998; 13: 521-530PubMed Google Scholar, 12Yin X.M. Oltvai Z.N. Korsmeyer S.J. Nature. 1994; 369: 321-323Crossref PubMed Scopus (1221) Google Scholar). Structural and mutational analyses have been used to assess the function of each BH domain in anti-apoptotic Bcl-2 family members. The BH4 domain, which is conserved only among anti-apoptotic Bcl-2 family members, has been reported to bind with other proteins regulating apoptosis, including calcineurin (13Shibasaki F. Kondo E. Akagi T. McKeon F. Nature. 1997; 386: 728-731Crossref PubMed Scopus (333) Google Scholar), Raf-1 (14Wang H.G. Rapp U.R. Reed J.C. Cell. 1996; 87: 629-638Abstract Full Text Full Text PDF PubMed Scopus (710) Google Scholar), and voltage-dependent anion channel (VDAC) (15Shimizu S. Narita M. Tsujimoto Y. Nature. 1999; 399: 483-487Crossref PubMed Scopus (1928) Google Scholar), and deletion of BH4 from Bcl-2/Bcl-xL has been shown to abrogate their anti-apoptotic ability (16Huang D.C.S. Adams J.M. Cory S. EMBO J. 1998; 17: 1029-1039Crossref PubMed Scopus (201) Google Scholar, 17Hunter J.J. Bond B.L. Parslow T.G. Mol. Cell. Biol. 1996; 16: 877-883Crossref PubMed Google Scholar, 18Borner C. Martinou I. Mattmann C. Irmler M. Schaerer E. Martinou J.C. Tschopp J. J. Cell Biol. 1994; 126: 1059-1068Crossref PubMed Scopus (175) Google Scholar, 19Hanada M. Aime-Sempe C. Sato T. Reed J.C. J. Biol. Chem. 1995; 270: 11962-11969Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar, 20Lee L.C. Hunter J.J. Mujeeb A. Turck C. Parslow T.G. J. Biol. Chem. 1996; 271: 23284-23288Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). This indicates that BH4 of anti-apoptotic Bcl-2 family members is crucial for anti-apoptotic events. The BH3 domain plays the role of ligand during dimerization of Bcl-2 family proteins, whereas a combination of the BH1, BH2, and BH3 domains appears to be required for forming an elongated hydrophobic cleft, which can bind the BH3-containing peptides of the death promoters (21Muchmore S.W. Sattler M. Liang H. Meadows R.P. Harlan J.E. Yoon H.S. Nettesheim D. Chang B.S. Thompson C.B. Wong S.L. Ng S.L. Fesik S.W. Nature. 1996; 381: 335-341Crossref PubMed Scopus (1289) Google Scholar, 22Sattler M. Liang H. Nettesheim D. Meadows R.P. Harlan J.E. Eberstadt M. Yoon H.S. Shuker S.B. Chang B.S. Minn A.J. Thompson C.B. Fesik S.W. Science. 1997; 275: 983-986Crossref PubMed Scopus (1301) Google Scholar, 23Reed J.C. Nature. 1997; 387: 773-776Crossref PubMed Scopus (1391) Google Scholar). Earlier studies have indicated that the BH1 and BH2 domains of Bcl-2/Bcl-xL are crucial for dimerization with pro-apoptotic family members (12Yin X.M. Oltvai Z.N. Korsmeyer S.J. Nature. 1994; 369: 321-323Crossref PubMed Scopus (1221) Google Scholar, 24Sedlak T.W. Oltvai Z.N. Yang E. Wang K. Boise L.H. Thompson C.B. Korsmeyer S.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7834-7838Crossref PubMed Scopus (784) Google Scholar). Through BH1 and BH2, anti-apoptotic proteins can heterodimerize with the pro-apoptotic protein, and the ratio between anti- and pro-apoptotic proteins determines survival or death following various apoptotic stimuli. Mutation of the highly conserved glycine 145 to alanine of the BH1 domain in Bcl-2 (Bcl-2/G145A) (12Yin X.M. Oltvai Z.N. Korsmeyer S.J. Nature. 1994; 369: 321-323Crossref PubMed Scopus (1221) Google Scholar), the homologous G138A mutation in Bcl-xL (Bcl-xL/G138A) (24Sedlak T.W. Oltvai Z.N. Yang E. Wang K. Boise L.H. Thompson C.B. Korsmeyer S.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7834-7838Crossref PubMed Scopus (784) Google Scholar), or tryptophan 188 to alanine of the BH2 domain in Bcl-2 abrogates the ability of Bcl-2 and Bcl-xL to dimerize with Bax. These mutations also fail to inhibit cell death induced by deprivation of interleukin-3 (IL-3) or dexamethasone in the murine prolymphocytic IL-3-dependent cell line, F5.12 (12Yin X.M. Oltvai Z.N. Korsmeyer S.J. Nature. 1994; 369: 321-323Crossref PubMed Scopus (1221) Google Scholar, 24Sedlak T.W. Oltvai Z.N. Yang E. Wang K. Boise L.H. Thompson C.B. Korsmeyer S.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7834-7838Crossref PubMed Scopus (784) Google Scholar). In addition, deletion of BH1 or BH2 has been shown to abrogate the death repressor activity of Bcl-2 in staurosporine-induced cell death in human GM701 fibroblasts (17Hunter J.J. Bond B.L. Parslow T.G. Mol. Cell. Biol. 1996; 16: 877-883Crossref PubMed Google Scholar). Contrary to these indications, others have suggested that heterodimerization of Bcl-2/Bcl-xL with Bax through the BH1 and BH2 domains is not required for anti-apoptotic function (25Cheng E.H. Levine B. Boise L.H. Thompson C.B. Hardwick J.M. Nature. 1996; 379: 554-556Crossref PubMed Scopus (444) Google Scholar, 26Kelekar A. Chang B.S. Harlan J.E. Fesik S.W. Thompson C.B. Mol. Cell. Biol. 1997; 17: 7040-7046Crossref PubMed Scopus (271) Google Scholar). Moreover, Oh et al. (27Oh Y.J. Uhland-Smith A. Kim J.E. O'Malley K.L. Mol. Brain Res. 1997; 51: 133-142Crossref PubMed Scopus (17) Google Scholar) has reported that overexpression of the BH1 or BH2 point mutant Bcl-2 protein suppressed staurosporine-induced apoptosis in a dopaminergic neuronal cell line, MN9D. Therefore, these contradictory findings suggest that the requirement of these dimerization domains of Bcl-2/Bcl-xL may be cell- and/or stress type-specific, and the mechanism of regulating apoptosis by Bcl-2 family member is not fully elucidated. In the course of examining the role of homo- and heterodimerization of Bcl-2 family members in antitumor drug-induced apoptosis using various Bcl-2 mutant-overexpressing human small cell lung carcinoma (SCLC) Ms-1 cells, we found that overexpression of Bcl-2 lacking a BH1 domain accelerated antitumor drug-induced apoptosis. Here, we show that a deletion mutant of the BH1 domain of Bcl-2 acts as a dominant negative of anti-apoptotic proteins of the Bcl-2 family. Materials—Camptothecin, colchicine, anti-α-tubulin (clone DM 1A) monoclonal antibody, and anti-Bak (B5929) antibody were purchased from Sigma Chemical Co. Vinblastine sulfate and paclitaxel were obtained from Wako Pure Chemical Industries, Ltd. (Tokyo, Japan). C2-ceramide was obtained from Biomol (Plymouth Meeting, PA). Anti-poly(ADP-ribose) polymerase (PARP) (H-250), anti-Bcl-2 (ΔC21), and anti-Bax (N-20) polyclonal antibodies and anti-Myc (9E10) monoclonal antibody were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-cytochrome c (clone 7H8.2C12) monoclonal antibody was obtained from BD PharMingen (San Diego, CA). Anti-Bcl-2 (clone 124) monoclonal antibody was from DAKO (Glostrup, Denmark). Plasmids—pGEM-T Easy-human bcl-2, -human bcl-xL, and -human bax plasmids and pCI-neo plasmid were kindly provided by Dr. S. Simizu (RIKEN). pCAGGS-human bak plasmid was kindly provided by Dr. Y. Tsujimoto (Osaka University Graduate School of Medicine, Japan). To generate the pCI-myc-neo plasmid, the fragment of 6× Myc epitope in pCS-MT was excised by digestion with BamHI/XbaI, blunted using 1 unit of T4 DNA polymerase (Takara, Tokyo, Japan) and then subcloned into an EcoRI-digested and T4 DNA polymerase-blunted pCI-neo plasmid. Bcl-2 deletion mutants were previously described (28Kawatani M. Uchi M. Simizu S. Osada H. Imoto M. Exp. Cell Res. 2003; (in press)PubMed Google Scholar). Bcl-2 point mutants, Bcl-2/G145A and Bcl-2/D140A, were constructed as follows. First, 100 ng of pGEM-T Easy-human bcl-2 plasmid was used for amplification under the following PCR conditions: 35 cycles of denaturation for 1 min at 94 °C, annealing for 1 min at 50 °C, and extension for 2 min at 72 °C. The following specific primers were used: Bcl-2/G145A mutant, 5′-GGGACGCTTTGCCACGGTGGTGGAGGAGCTCTTCAGGGACGGGGTGAACTGGGCAAGGATTGTGGCCTTC-3′ (forward) and 5′-TATTTAGGTGACACTATAG-3′ (SP-6, reverse); Bcl-2/D140A mutant, 5′-GGGACGCTTTGCCACGGTGGTGGAGGAGCTCTTCAGGGCAGG GGTGAACTGGGGGAGG-3′ (forward) and 5′-TATTTAGGTGACACTATAG-3′ (SP-6, reverse). These PCR products were digested by BstXI, and then subcloned into a BstXI-digested pGEM-T Easy-human bcl-2 plasmid. These constructs were confirmed by DNA sequence analysis and digested by EcoRI, and then cloned into the mammalian expression plasmid, pCI-neo or pCI-myc-neo. Cell Culture and Transfection—Human SCLC Ms-1 and human embryonic kidney 293T cells were cultured in RPMI 1640 medium containing 5% fetal bovine serum, penicillin G (100 units/ml), and kanamycin (0.1 mg/ml) at 37 °C in 5% CO2, 95% air. For establishing stable cell lines, Ms-1 cells (2 × 106) were transfected with 10 μg of plasmid pCI-neo alone or pCI-neo containing various constructs using LipofectAMINETM reagent (Invitrogen) according to the manufacturer's instructions. These transfectants were selected by supplementing the medium with 700 μg/ml G418 (Sigma Chemical Co.). Single cell clones were isolated from the bulk transfectants by limited dilution cloning in 96-well plates and characterized by Western blot analysis as described below. 293T cells were transiently transfected with 1 μg of plasmid DNA per 105 cells using the LipofectAMINETM reagent according to the manufacturer's instructions. Western Blot Analysis—The cells were lysed in radioimmune precipitation assay buffer (25 mm HEPES, 1.5% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 0.5 m NaCl, 5 mm EDTA, 50 mm NaF, 0.1 mm sodium vanadate, 1 mm phenylmethylsulfonyl fluoride, and 0.1 mg/ml leupeptin, pH 7.8) at 4 °C with sonication. The lysates were centrifuged at 15,000 × g for 15 min, and the concentration of the protein in each lysate was determined with Coomassie Brilliant Blue G-250. Loading buffer (42 mm Tris-HCl, 10% glycerol, 2.3% SDS, 5% 2-mercaptoethanol, and 0.002% bromphenol blue) was then added to each lysate, which was subsequently boiled for 3 min and then electrophoresed on an SDS-polyacrylamide gel. Proteins were transferred to Hybond-P membrane (Amersham Biosciences) and immunoblotted with anti-Bcl-2 (clone 124 or ΔC21), anti-Bax (N-20), anti-PARP (H-250), anti-Bak (B5929) or anti-Myc (9E10) antibody. Detection was performed with enhanced chemiluminescence reagent (PerkinElmer Life Sciences). Cell Viability Assay—Drug-treated or untreated Ms-1 cells or transiently transfected 293T cells were stained with trypan blue (Sigma Chemical Co.), and the percentage of viable cells was determined using a hemocytometer. Cell viability (%) means the ratio of the number of trypan blue-impermeable cells in total cell counts (trypan blue-impermeable cell number/total cell number). Apoptosis Assay—Cells were seeded onto glass coverslips. Detection of apoptotic cells were performed by annexin V and propidium iodide (PI) staining using the annexin V-EGFP apoptosis detection kit (MBL, Nagoya, Japan) according to the manufacturer's instructions. Apoptotic cells (annexin V-positive and PI-negative cells) were scored under a fluorescence microscope. Evaluation of Cytosolic Cytochrome c—Drug-treated or untreated cells (1 × 107) were harvested and washed with ice-cold phosphate-buffered saline. Cells were resuspended in 600 μl of buffer A (20 mm HEPES-KOH, pH 7.5, 10 mm KCl, 1.5 mm MgCl2, 1 mm EDTA, 1 mm EGTA, and 1 mm dithiothreitol) containing 250 mm sucrose and a mixture of protease inhibitors (1 mm phenylmethylsulfonyl fluoride, 1% aprotinin, 1 mm leupeptin, 1 μg/ml pepstatin A, and 1 μg/ml chymostatin). The cells were homogenized with 20 strokes of a glass Pyrex homogenizer. Unbroken cells, large plasma membrane pieces, and nuclei were removed by centrifuging the homogenates at 1,000 × g for 10 min at 4 °C. The supernatant was centrifuged at 100,000 × g for 1 h at 4 °C to prepare cytosol. The resulting supernatant was used as the soluble cytosolic fraction. Western blot analysis for cytochrome c (anti-cytochrome c (clone 7H8.2C12) monoclonal antibody) or α-tubulin (anti-α-tubulin (clone DM 1A) monoclonal antibody) was performed. Immunoprecipitation—Transiently transfected 293T cells were resuspended in Extraction buffer (50 mm HEPES, 150 mm NaCl, 1 mm EDTA, 2.5 mm EGTA, 1 mm dithiothreitol, 0.1% Tween 20, 10 mm sodium-β-glycerophosphate, 10% glycerol, 1 mm NaF, 0.1 mm sodium vanadate, 1 mm phenylmethylsulfonyl fluoride, and 0.1 mg/ml leupeptin, pH 7.5), sonicated on ice and centrifuged at 15,000 × g for 20 min at 4 °C. The supernatants were precleared by using protein G-agarose resin suspension (Oncogene Research Products, Boston, MA), and then immunoprecipitated using 10 μl of anti-Bax (N-20), anti-Bak (B5929), or anti-Myc (9E10) antibody overnight at 4 °C. Next, 10 μl of protein G-agarose resin suspension was added to each of the immunocomplexes, and the mixtures were rotated for 5 h at 4 °C. After 4 washes with Extraction buffer, the immunocomplexes were eluted with SDS sample buffer. The immunoprecipitated proteins were subjected to SDS-PAGE and Western blot analysis with the antibodies. Overexpression of Bcl-2/ΔBH1 Accelerates Camptothecin-induced Apoptosis in Ms-1 Cells—Stable clones of human SCLC Ms-1 cells, overexpressing wild-type (wt) Bcl-2, lacking the BH1 domain of Bcl-2 (Bcl-2/ΔBH1) or vector only (neo), were exposed to camptothecin, and their cell viabilities were assessed by trypan blue dye exclusion assay. As shown in Fig. 1A, camptothecin decreased the viability of vector control clone (Ms-1/neo 13) in a time-dependent manner and failed to induce cell death in the wt Bcl-2-overexpressing Ms-1 clone (Ms-1/wt Bcl-2 7). On the other hand, camptothecin-induced cell death was markedly accelerated in the Bcl-2/ΔBH1-overexpressing Ms-1 clone (Ms-1/Bcl-2/ΔBH1 72) when compared with the Ms-1/neo 13 clone (Fig. 1A). When Ms-1/Bcl-2/ΔBH1 72 was treated with 0.1 or 0.3 μg/ml camptothecin for 18 h, cell viability decreased about 60 or 40%, respectively, under the conditions where significant cell death was not observed in Ms-1/neo 13 (Fig. 1B), indicating that expression of Bcl-2/ΔBH1 reduced the dosage of camptothecin required to induce cell death. Similar results were obtained from independently derived clones (Fig. 1C). Moreover, overexpression of Bcl-2/ΔBH1 also accelerated cell death induced by vinblastine, paclitaxel, or colchicine, as shown in Fig. 1D, suggesting that the acceleration of cell death by Bcl-2/ΔBH1 is not specific to camptothecin-induced cell death. On the other hand, Bcl-2/ΔBH1 and wt Bcl-2 inhibited C2-ceramide-induced cell death (Fig. 1D) as well as cytochrome c release and caspase-3(-like) protease activation (data not shown). Bcl-2/ΔBH1, but Not Bcl-2/ΔBH2, Bcl-2/ΔBH3, and Bcl-2/ΔBH4, Accelerates the Step of Cytochrome c Release from Mitochondria Induced by Camptothecin—Next, we examined whether other BH domain deletion mutants of Bcl-2/ΔBH2, Bcl-2/ΔBH3, and Bcl-2/ΔBH4 also accelerated the cell death induced by camptothecin. As shown in Fig. 2, although all deletion mutant clones tested expressed similar amounts of mutant Bcl-2 protein (Fig. 2A), significant cell death was observed in the Ms-1/Bcl-2/ΔBH1 no. 72 clone but not the Ms-1/Bcl-2/ΔBH2 no. 109, Ms-1/Bcl-2/ΔBH3 no. 55, or Ms-1/Bcl-2/ΔBH4 no. 35 clone 10 h following camptothecin treatment (Fig. 2B). Fig. 2C shows that 18 h after treatment with 1 μg/ml camptothecin, the cell viability of the Ms-1/Bcl-2/ΔBH3 no. 55 and Ms-1/Bcl-2/ΔBH4 no. 35 clones was reduced, and the rate of cell death in these clones was quite similar to that in Ms-1/neo no. 13; however, the Ms-1/Bcl-2/ΔBH2 no. 109 clone was resistant to camptothecin. Similar results were obtained from at least two independently derived clones from each transfectant (data not shown). We next examined the effect of Bcl-2/ΔBH1 on camptothecin-induced cytochrome c release. As shown in Fig. 3A, cytosolic cytochrome c was detected in Ms-1/Bcl-2/ΔBH1 no. 72 at 1 h after camptothecin treatment, under the conditions where cytochrome c release was not yet detected in camptothecin-treated Ms-1/neo no. 13 or the other transfectants. Moreover, at 2 h after the treatment with camptothecin, activation of caspase-3(-like) proteases, as estimated by the cleavage of PARP, was also markedly induced only in the Ms-1/Bcl-2/ΔBH1 no. 72 clone (Fig. 3B). These results indicate that Bcl-2/ΔBH1 accelerates the release of cytochrome c leading to caspase-3-(-like) protease activation induced by camptothecin, thereby enhancing the cell death. On longer exposure to camptothecin, the release of cytochrome c (4 h) and cleavage of PARP (6 h) occurred in Ms-1/Bcl-2/ΔBH3 no. 55 and Ms-1/Bcl-2/ΔBH4 no. 35 as well as in Ms-1/neo no. 13 (Fig. 3, A and B). In contrast, the Ms-1/Bcl-2/ΔBH2 no. 109 clone showed significant resistance to camptothecin, when compared with the Ms-1/neo no. 13 clone (Fig. 3, A and B). Mutant Bcl-2/G145A but Not Bcl-2/D140A Accelerates Camptothecin-induced Cell Death—Mutational and structural analyses have indicated that the BH1 domain of Bcl-2/Bcl-xL is crucial for dimerization with pro-apoptotic family members (12Yin X.M. Oltvai Z.N. Korsmeyer S.J. Nature. 1994; 369: 321-323Crossref PubMed Scopus (1221) Google Scholar, 25Cheng E.H. Levine B. Boise L.H. Thompson C.B. Hardwick J.M. Nature. 1996; 379: 554-556Crossref PubMed Scopus (444) Google Scholar). Therefore, to investigate whether the promotional effect on apoptosis of Bcl-2/ΔBH1 was due to a failure of interaction with pro-apoptotic members, Bax and Bak, we generated two point mutations in the BH1 domain of Bcl-2: Bcl-2/G145A, which had been unable to bind Bax or Bak (12Yin X.M. Oltvai Z.N. Korsmeyer S.J. Nature. 1994; 369: 321-323Crossref PubMed Scopus (1221) Google Scholar); Bcl-2/D140A, which corresponds with Bcl-xL/D133A that had been able to bind Bak, but not Bax (25Cheng E.H. Levine B. Boise L.H. Thompson C.B. Hardwick J.M. Nature. 1996; 379: 554-556Crossref PubMed Scopus (444) Google Scholar). Each of these mutant Bcl-2 products was first tested for the ability to heterodimerize with Bax or Bak. 293T cells were transiently co-transfected with pCI-neo plasmids encoding Bax or Myc-tagged Bak and wt Bcl-2 or these mutants, and co-immunoprecipitation was performed with anti-Bax or anti-Myc antibody. In agreement with a previous report (12Yin X.M. Oltvai Z.N. Korsmeyer S.J. Nature. 1994; 369: 321-323Crossref PubMed Scopus (1221) Google Scholar), Bcl-2/G145A bound neither Bax nor Myc-tagged Bak (Fig. 4, A and B). We found that Bcl-2/D140A failed to interact with Bax, but still interacted with Myc-tagged Bak (Fig. 4, A and B). Then, we established stable Ms-1 cells overexpressing Bcl-2/G145A or Bcl-2/D140A (Fig. 5A) and analyzed them for the ability to enhance camptothecin-induced cell death. Two clones overexpressing Bcl-2/G145A (Ms-1/Bcl-2/G145A nos. 1 and 4) displayed increased camptothecin-induced cell death compared with Ms-1/neo no. 13 with similar kinetics observed in Ms-1/Bcl-2/ΔBH1 no. 72, as shown in Fig. 5B. In contrast, camptothecin-induced cell death was completely inhibited in two clones overexpressing Bcl-2/D140A (Ms-1/Bcl-2/D140A nos. 25 and 17) (Fig. 5C). Similarly, Ms-1/Bcl-2/G145A no. 4 displayed increased cytosolic cytochrome c and apoptosis induced by camptothecin compared with Ms-1/neo no. 13, however, Ms-1/Bcl-2/D140A no. 25 as well as Ms-1/wt Bcl-2 no. 7 was resistant to camptothecin (Fig. 5, D and E). Thus, Bcl-2/G145A but not Bcl-2/D140A showed similar activity to Bcl-2/ΔBH1, suggesting that the promotional effect on apoptosis of Bcl-2/ΔBH1 might require Bcl-2 to lose the interaction with both Bax and Bak. On the other hand, cytochrome c release and apoptosis induced by C2-ceramide were not observed in Ms-1/Bcl-2/G145A no. 4 and Ms-1/Bcl-2/D140A no. 25 (Fig. 5, D and E).Fig. 5Overexpression of Bcl-2/G145A but not Bcl-2/D140A accelerates camptothecin-induced apoptosis in Ms-1 cells.A, Western blot analysis of wt Bcl-2 and BH1 domain point mutanto-verexpressing Ms-1 cell lines. Equal aliquots of protein extracted from stable transfectants of the indicated Ms-1 cells were immunoblotted with anti-Bcl-2 (clone 124) or anti-Bax antibody. B, stable transfectants of the indicated Ms-1 cells were treated with 1 μg/ml camptothecin for the periods indicated. C, stable transfectants of the indicated Ms-1 cells were treated with 1 μg/ml camptothecin for 18 h. Cell viability was assessed by trypan blue dye exclusion assay. Values are means ± S.D. of quadruplicate determinations. D, stable transfectants of the indicated Ms-1 cells were treated with 1 μg/ml camptothecin for 8 h or 30 μm C2-ceramide for 8 h. Apoptotic cells (annexin V-positive and PI-negative cells) were scored under a fluorescence microscope. Values are means ± S.D. of quadruplicate determinations. E, stable transfectants of the indicated Ms-1 cells were treated with 1 μg/ml camptothecin for 4 h or 30 μm C2-ceramide for 6 h. Cytosolic proteins were isolated and immunoblotted with anti-cytochrome c or anti-α-tubulin antibody, as described under “Experimental Procedures.”View Large Image Figure ViewerDownload Hi-res image Download (PPT) The BH2 domain as well as BH1 domain of Bcl-2/Bcl-xL has been reported to be crucial for dimerization with pro-apoptotic family members (12Yin X.M. Oltvai Z.N. Korsmeyer S.J. Nature. 1994; 369: 321-323Crossref PubMed Scopus (1221) Google Scholar, 25Cheng E.H. Levine B. Boise L.H. Thompson C.B. Hardwick J.M. Nature. 1996; 379: 554-556Crossref PubMed Scopus (444) Google Scholar). However, overexpression of Bcl-2/ΔBH2 did not accelerate camptothecin-induced apoptosis, but rather, significantly inhibited it (Figs. 2 and 3). In order to verify this difference, we confirmed the interaction between Bcl-2/ΔBH2 and Bax or Bak. The pCI-neo plasmid encoding wt Bcl-2, Bcl-2/ΔBH1, Bcl-2/G145A, or Bcl-2/ΔBH2 was transiently co-transfected with the pCI-neo plasmid encoding Bax or Myc-tagged Bak into 293T cells and co-immunoprecipitation was performed with anti-Bax or anti-Myc antibody. As shown in Fig. 6A and B, although the binding activity was reduced, Bcl-2/ΔBH2 still retained the ability to bind to Bax or Myc-tagged Bak, under the conditions where Bcl-2/ΔBH1 or Bcl-2/G145A failed to interact with Bax or Myc-tagged Bak, respectively. Therefore, these results suggest that the different activity between Bcl-2/ΔBH1 and Bcl-2/ΔBH2 for regulating apoptosis induced by camptothecin is caused by the difference between Bcl-2/ΔBH1 and Bcl-2/ΔBH2 in interacting with pro-apoptotic members, Bax and Bak. Bcl-2/G145A as Well as Bcl-2/ΔBH1 Accelerates Bax- or Bak-induced Apoptosis—Because Bcl-2/G145A and Bcl-2/ΔBH1 failed to bind to Bax and Bak, we next examined whether Bax- or Bak-induced apoptosis is also accelerated by these two mutants. The pCI-neo plasmid encoding wt Bcl-2 or Bcl-2/G145A, or control vector was transfected into 293T cells with or without the pCI-neo plasmid encoding Bax. As shown in Fig. 7A, expression of Bax induced cell death in a time-dependent manner, and wt Bcl-2 completely blocked the Bax-induced cell death. In contrast, Bcl-2/G145A significantly accelerated Bax-induced cell death (Fig. 7A). A similar result was obtained when cells were transfected with Bak instead of Bax (Fig. 7B). Acceleration of Bax- or Bak-induced cell death was also observed by Bcl-2/ΔBH1 expression (Fig. 7, C and D). Moreover, Bcl-2/G145A accelerated Bax-induced caspase-3(-like) protease activation under the conditions where wt Bcl" @default.
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• W2023973334 title "Deletion of the BH1 Domain of Bcl-2 Accelerates Apoptosis by Acting in a Dominant Negative Fashion" @default.
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